Method of obtaining antibodies

Abstract

Antibodies specific to glycosylated hemoglobin and substantially free of cross-reactivity with nonglycosylated forms of hemoglobin are obtained by administering a synthetic antigen to a host animal. The antigen comprises a glycosylated synthetic peptide having an amino acid sequence corresponding to that of the NH 2 -terminus of the β-chain of hemoglobin. The peptide comprises a sequence of between about four and ten, and preferably seven, amino acids, and is conjugated with glucose and a protein. The protein preferably comprises an immunoglobulin which is foreign to the host animal.

We claim: 
     
       1. A method of preparing an antigen useful in obtaining antibodies against HbAlc, which antibodies are substantially free of cross-reactivity to nonglycosylated hemoglobin, said method comprising the steps of: (a) synthesizing a hapten consisting of a peptide having the formula Val--His--Leu--Y wherein Y is chosen from the group consisting of Thr--, Thr--Pro--, Thr--Pro--Glu--, Thr--Pro--Glu--Glu--, Thr--Pro--Glu--Glu--Lys--, Thr--Pro--Glu--Glu--Lys--Ser--, and Thr--Pro--Glu--Glu--Lys--Ser--Ala--, said hapten thereby having an amino acid sequence corresponding to the NH 2  terminus of the hemoglobin β-chain;   (b) reacting the peptide of step (a) with glucose to obtain a glycosylated peptide having the formula   glucose--Val--His--Leu--Y;        and   (c) conjugating the glycosylated peptide of step (b) with a protein to form an antigen suitable for injection into a host animal whose metabolism does not naturally produce HbAlC for production of antibodies thereto.   
     
     
       2. The method of claim 1 wherein said protein is an immunoglobulin. 
     
     
       3. The method of claim 2 wherein said protein comprises IgG. 
     
     
       4. The method of claim 1 wherein said peptide is covalently linked to said protein. 
     
     
       5. The method of claim 4 wherein said peptide is conjugated to said protein through a carbodiimide linkage. 
     
     
       6. The method of claim 2 wherein said peptide comprises d-glucose--Val--His--Leu--Thr--Pro--Glu--Glu--, said protein is bovine IgG, and said peptide is covalently linked to said bovine IgG through a carbodiimide linkage.